Induction Of Intracellular Signals Research Articles

Bone Marrow Stromal Antigen 2 Facilitates Activation of Hematopoietic Stem Cell through the Induction of ERK Signaling

Chronic infections are prevalent worldwide and contribute to bone marrow failure syndromes. Long-term homeostatic production of all blood cells is dependent on the ability of hematopoietic stem cells (HSCs) to remain mostly quiescent and, upon activation, must carefully balance between their ability to self-renew and differentiate. However, infections trigger inflammatory cytokines, such as interferon-gamma (IFNy), that facilitate HSC proliferative exhaustion by disrupting both quiescence and self-renewal, thereby contributing to cytopenias and bone marrow failure syndromes. Our previously published results indicate that the IFN-induced surface protein, Bone Marrow Stromal Antigen 2 (BST2), facilitates HSC activation, and the loss of BST2 protected against the depletion of HSCs during chronic infection. However, the mechanism by which BST2 impacts HSCs activation is unknown. BST2 is known to promote activation of intracellular signaling in both dendritic cells and cancer cell types. In particular BST2 is a known activator of the NFkB signaling pathway. We hypothesized that BST2 could promote activation of HSCs through the induction of intracellular signaling pathways.Intracellular signaling pathways have been shown to be important for the activation of not only HSCs but also downstream immune cells. Indeed, the loss of BST2 has been shown to impair the function of T cells, NK cells, and antigen presenting cells in the presence of viral infections via impaired induction of NFkB signaling. However, there have been no studies on the role of BST2 during bacterial infections. To evaluate the role of BST2 in hematopoiesis during a bacterial infection, we obtained BST2KO mice and characterized hematopoietic populations in the presence or absence of Mycobacterium avium infection, a model bacterial infection that generates high levels of IFNy. We observed no changes in activation or production of cytokines from macrophage, NK, CD8, or neutrophil populations. Surprisingly we observed enhanced dendritic cell activation at day 15, 30, and 42 as measured by co-stimulatory molecules and MHC II expression. Additionally, we observed increased tetramer-specific T cell numbers early on during the infection. Interestingly, these results pair with decreased bacterial load at day 30 and day 42 despite having the similar spleen sizes. These results suggest that BST2 regulates the activation of dendritic cells, which may trigger more robust earlier T cell responses early in the course of infection.Our previous work showed that the loss of BST2 protects against HSC activation by displacement of HSCs from CXCL12-abundant reticular (CAR) cells in the niche. To determine whether HSC activation is related to distancing from the vascular niche or increased proximity to an activated osteoblastic niche, we performed intravital imaging using osteocalcin-GFP reporter mice. We observed no change in proximity to the osteoblastic niche after administration of recombinant IFNy using either WT or BST2KO HSCs. These results suggest that the effects of BST2 may be more related to distancing from CAR cells than migration towards other niche locations.To evaluate the mechanisms underlying BST2-dependent HSC activation, we performed RNA sequencing on WT versus BST2KO HSCs in the presence of IFNy. GSEA-pathway analysis of differentially expressed genes revealed that BST2KO HSCs have a decreased response to IFNy and diminished activation of the P13K-AKT-MTOR signaling pathways. Western blot analysis on hematopoietic stem cell progenitor cells (HSPCs) indicate a decrease in phosphorylation of the ERK signaling pathway in BST2KO HSPCs. These results were analogous to work performed in 32D cells in vitro, in which ERK-phosphorylation has been shown to be important for both HSC maintenance and proliferation. Future studies are aimed at assessing the impact of ERK inhibition on HSC activation during various inflammatory stresses.Collectively our results show that the loss of BST2 in downstream immune cells surprisingly enhances dendritic cell and T cell activation in response to bacterial infection. BST2 enhances HSC activation through activation of downstream ERK signaling. Future targeting of BST2 and/or ERK signaling could have important consequences for patients that suffer from cytopenias, bone marrow failure, and engraftment failure due to high levels of inflammatory stimuli. DisclosuresNo relevant conflicts of interest to declare.

Establishment of Structure-Function Relationship of Tissue Inhibitor of Metalloproteinase-1 for Its Interaction with CD63: Implication for Cancer Therapy

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a pleiotropic protein, promoting both tumor-suppressive and tumor-promoting activities. While TIMP-1 is primarily known as an endogenous inhibitor of matrix metalloproteinases (MMPs) and thus associated with tumor cell invasion, clinical studies demonstrated increased expression of TIMP-1 and its association with poor prognosis in cancer. Non-MMP-inhibitory and oncogenic functions of TIMP-1 are mediated by induction of intracellular signaling via its cell surface receptor CD63, a tetraspanin. The present study investigates the structure-function relationship of TIMP-1 for its interaction with CD63, which may eventually help design a novel approach for targeting TIMP-1’s pro-oncogenic activity without interfering its tumor suppressive MMP-inhibitory function. Importantly, our analysis includes TIMP-1/CD63 interactions at the cell surface of live cells. Here, we demonstrate that the 9 C-terminal amino acid residues of TIMP-1 and the large extracellular loop of CD63 are required for their interaction. Considering that the N-terminal half of TIMP-1 is sufficient for TIMP-1’s MMP-inhibitory activity, we propose that those C-terminal amino acid residues are a potentially targetable motif of TIMP-1 oncogenic activity. As a proof of concept, we present the potential for the development of neutralizing antibodies against the C-terminal motif of TIMP-1 for disruption of TIMP-1 interaction with CD63 and the subsequent signal transduction.

Experimental Infection by Brucella ovis: Changes in NTPDase, 5'-Nucleotidase and Acetylcholinesterase Associated Cerebral Oxidative Stres

Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associated with inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are not known. B. ovis is generally asymptomatic in sheep. Hepatosplenomegaly has been described in B. ovis, a non-zoonotic species, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved with the modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigate the role of ectonucleotidases and cholinesterase’s in the brains of mice experimentally infected with B. ovis.Materials, Methods & Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24). In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brains were collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substances reactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP and ADP as substrate) and 5'-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathological analysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI. However,multifocal areas with moderate microgliosis and inflammatory infiltrates in the cerebral cortex were observed at day 60 PI in the infected animals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation in the brains of infected animals than in the control group at day 60PI. No significant results were observed at 7, 15 or day 30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than in the control group. NTPDase activity (using ATP and AMP as substrate) was lower at days 7 and 15 PI in infected animals than in control. However, during the course of infection there was an increase in NTPDase activity at day 60 PI in the infected group. The infected animals showed a decrease of 5´-nucleotidase (AMP as substrate) activity at days 7 and 30 PI. On the other hand, 5´-nucleotidase activity was greater on day 60 PI in the experimental group than in the control. The results suggest that nucleotide hydrolysis was low in the acute phase (up to day 30 PI) due to the decrease of NTPDase and 5´-nucleotidase activities. After day 60 PI, there was a reversal in enzyme activities, probably with concomitant increase of extracellular nucleotides. AChE activity in brain on days 30 and 60 PI compared to control.Discussion: Among the functions of NTPDase are inhibition of platelet aggregation, vascular homeostasis, modulation of inflammation and immune response, all via its regulation of extracellular concentrations of ATP, a pro-inflammatory molecule. E-NTPDase plays an important role in controlling lymphocyte function, including antigen recognition and activation of cytotoxic T cell effector functions, as well as the generation of signals. The enzyme E-5´-nucleotidase also exerts non-enzymatic functions, including induction of intracellular signaling and mediation of cell-cell adhesion and cell-matrix and migration. Levels of acetylcholine are regulated by cholinesterase enzymes that are present in cholinergic and noncholinergic tissues, as the acetylcholinesterase (AChE) is a membrane-bound enzyme, primarily found in the brain and cholinergic neurons, where it participates in the structural regulation of postsynaptic differentiation. The results demonstrated that the chronicity of infection by B. ovis causes oxidative damage and inflammation in the brain, as well as modulation of ectonucleotidases and AChE activities.

MP34-05 THE OXIDATIVE STRESS RESPONSE OF UROTHELIAL CARCINOMA CELLS TO BCG EXPOSURE

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2014MP34-05 THE OXIDATIVE STRESS RESPONSE OF UROTHELIAL CARCINOMA CELLS TO BCG EXPOSURE Gopitkumar Shah, Fanghong Chen, Gina Lockwood, Scott Johnson, Jacek Zielonka, Balaraman Kalyanaraman, and William See Gopitkumar ShahGopitkumar Shah More articles by this author , Fanghong ChenFanghong Chen More articles by this author , Gina LockwoodGina Lockwood More articles by this author , Scott JohnsonScott Johnson More articles by this author , Jacek ZielonkaJacek Zielonka More articles by this author , Balaraman KalyanaramanBalaraman Kalyanaraman More articles by this author , and William SeeWilliam See More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1018AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Earlier studies have shown that the cellular oxidative stress (COS), post BCG treatment plays a central role in mediating direct effect of BCG on urothelial carcinoma (UC) cells. The recent novel findings that BCG acts as an H2O2 generator provides insight into the genesis of BCG induced COS. To date the net impact of BCG exposure on specific contributors to COS remains poorly defined. The present study reports the results of experiments designed to assess the broad impact of BCG on inducers and regulators of COS as well as downstream “targets” of COS. METHODS Global profiling of reactive oxygen (ROS) and nitrogen species (RNS) was carried out in two human UC cell lines (253J, T24) using fluorescent probes for H2O2, nitric oxide (NO), and superoxides at various time points post BCG exposure. Enzymatic regulators of COS, Superoxide dismutase (SOD), catalase and Glutathione peroxidase (GPx) activities were measured at 6h post BCG exposure. Downstream targets of COS, DNA damage and lipid peroxidation, were determined by measuring 8-hydroxydeoxyguanosine (OHdG) and 4-hydroxynonenal (HNE) adduct, respectively. RESULTS Post BCG treatment, an increase in cellular H2O2 was observed as early as 3h (p ≤ 0.05) and a sustained increase was observed till 12h (p ≤ 0.005). H2O2 levels did not change significantly 24h onwards (p = 0.32). NO levels showed a significant increase at 6h (p ≤ 0.05) but not at 3h (p = 0.2) or 9h (p = 0.14) onwards. Superoxide levels showed significant increase at 6h (p ≤ 0.05) but reduced levels at 24h (p ≤ 0.05). Post BCG exposure, SOD and GPx activities were significantly higher at 6h (p ≤ 0.05), while catalase activity did not show any significant change (p = 0.5) in both cell lines. BCG treatment significantly increased lipid peroxidation (p ≤ 0.001) and DNA damage (p ≤ 0.05) in UC cells after 72h. Addition of iNOS inhibitor 1400W nullified the increase in NO, lipid peroxidation and DNA damage. CONCLUSIONS Exposure of UC cells to BCG initiates a temporally complex COS response in the treated cells. Early production of H2O2 post BCG treatment suggests that H2O2 acts to initiate a primary wave of oxidative stress. Continued production of H2O2 leads to induction of NO and superoxide as a second wave of COS which leads to lipid peroxidation and DNA damage. NO has been shown to play a a direct role in the induction of intracellular signaling, gene expression and phenotypic changes including cellular necrosis and release of HMGB1. COS appears to play a central role in mediating the direct effects of BCG on UC cell biology. COS is a candidate for targeted manipulation with the intent of increasing the efficacy of BCG. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e363 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Gopitkumar Shah More articles by this author Fanghong Chen More articles by this author Gina Lockwood More articles by this author Scott Johnson More articles by this author Jacek Zielonka More articles by this author Balaraman Kalyanaraman More articles by this author William See More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Induction of Phagocytosis and Intracellular Signaling by an Inhibitory Channel Catfish Leukocyte Immune-Type Receptor: Evidence for Immunoregulatory Receptor Functional Plasticity in Teleosts

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

Toll-like Receptor 2 Is Required for LPS-induced Toll-like Receptor 4 Signaling and Inhibition of Ion Transport in Renal Thick Ascending Limb

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) absorption by lumen LPS was preserved in TLR2(-/-) MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2(-/-) mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2(-/-) MTALs. Inhibition of HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.

Developmental changes of TrkB signaling in response to exogenous brain‐derived neurotrophic factor in primary cortical neurons

Neocortical circuits are most sensitive to sensory experience during a critical period of early development. Previous studies implicate that brain-derived neurotrophic factor (BDNF) and GABAergic inhibition may control the timing of the critical period. By using an in vitro maturation model, we found that neurons at DIV (day in vitro) 7, around a period when functional synapses start to form and GABAergic inhibition emerges, displayed the most dynamic activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB by exogenous BDNF. The BDNF-stimulated transcriptional up-regulation of CREB target genes was also the highest in DIV 7 neurons. The basal level of ERK1/2 and CREB activity, as well as the expression of CREB target genes, increased along with maturation, and neurons at DIV 13 and 22 displayed less dynamic responses to BDNF. Furthermore, we found that the developmentally regulated GABAergic inhibition correlated with the decline of BDNF-mediated signaling during maturation. BDNF stimulation along with suppression of GABAergic inhibition enhanced the activation of ERK1/2-CREB signaling and gene transcription in mature neurons. Conversely, BDNF stimulation along with enhancement of GABAergic inhibition reduced the overall induction of intracellular signaling in younger neurons. We propose that the less dynamic molecular changes may play a certain role in the loss of plasticity during maturation.

Initiation of Plasmodium sporozoite motility by albumin is associated with induction of intracellular signalling

Malaria infection is initiated when a mosquito injects Plasmodium sporozoites into a mammalian host. Sporozoites exhibit gliding motility both in vitro and in vivo. This motility is associated with the secretion of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Both derive from micronemes, which are organelles that empty out of the apical end of the sporozoite. Sporozoite motility can be initiated in vitro by albumin added to the medium. To investigate how albumin functions in this process, we studied second messenger signalling within the sporozoite. Using pharmacological activators and inhibitors, we have concluded that gliding motility is initiated when albumin interacts with the surface of the sporozoite and that this leads to a signal transduction cascade within the sporozoite, including the elevation of intracellular cAMP, the modulation of sporozoite motility by Ca 2+ and the release of microneme proteins.

Induction of intracellular signalling in human endothelial cells by the hyaluronan-binding protease involves two distinct pathways

Recently a novel plasma serine protease with high affinity to hyaluronic acid and glycosaminoglycans, such as heparin and heparan sulfate, has been described and termed hyaluronan-binding protease (HABP). HABP cleaves kininogen in vitro, releasing the vasoactive peptide bradykinin, and activates plasminogen activators, suggesting a vascular cell-directed physiological function of this novel plasma protease. Here we show that HABP stimulates human umbilical vein endothelial cells (HUVECs) by activating two distinct cell-surface receptors. On the one hand, HABP releases bradykinin from cell surface-bound or soluble kininogen and triggers a bradykinin B2-receptor-dependent mobilisation of intracellular Ca2+. On the other hand, HABP activates the p44/42-dependent MAPK (ERK1/2) signalling cascade independent of the B2-receptor, but involving the fibroblast growth factor receptor-1 and basic fibroblast growth factor. This signalling pathway leads to phosphorylation of the kinases Raf, MEK1/2 and ERK1/2. The extracellular activity of HABP also affects the gene expression level through phosphorylation of two transcription factors, the cAMP-responsive element binding protein CREB and the proto-oncogene c-Myc. Our results indicate a proangiogenic potential of HABP, which, in combination with a profibrinolytic activity, directs the physiological function of this plasma protease to processes in which clot lysis, cell motility and neovascularisation are pivotal processes, e.g., in wound healing, tissue repair and tumour progression.

The signaling pathways induced by neutrophil-endothelial cell adhesion.

Adhesion of neutrophils to vascular endothelial cells (ECs), mediated by the interaction of CD11/CD18 and intercellular adhesion molecule-1 (ICAM-1), is often required for neutrophil transmigration across endothelium during most inflammatory responses. Induction of intracellular signaling in neutrophils as a result of adhesion has been recognized for many years. Recent studies demonstrated that neutrophil-endothelial adhesion also activates ECs. Examples of neutrophil adherence-induced changes in ECs include increases in intracellular Ca(2+), production of reactive oxygen species, and actin cytoskeleton changes. These changes result, in part, from ligation of EC adhesion molecules. This review article focuses on the signaling events that occur in ECs during neutrophil adhesion and the role of EC adhesion molecules, particularly ICAM-1, in the initiation of these signaling events in ECs. The evidence to date describing the molecular basis of ICAM-1-induced signaling will be summarized. Finally, the potential physiological roles of these signaling events induced by EC adhesion molecules in mediating neutrophil migration will be addressed.

Okadaic acid stimulates apoptosis through expression of Fas receptor and Fas ligand in human oral squamous carcinoma cells.

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to Fas receptor leads to activation of the latter and the induction of intracellular signals that result in apoptotic cell death. In the present study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to examine the expression of mRNAs and proteins of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. The PCR product of Fas receptor mRNA was detected in the cells and a protein with an estimated molecular weight of 35,000 was also expressed in them. Expression of Fas receptor mRNA stimulated by okadaic acid was elevated in dose- and time-dependent manners as judged by semiquantitative RT-PCR analysis, with the maximum expression level at 50 nM and 8 h treatment. Fas ligand mRNA expression was also stimulated by okadaic acid in SCC-25 cells in dose- and time-dependent manners. Okadaic acid also stimulated the expression of Fas ligand protein in the cells. Okadaic acid in serum-free medium induced apoptosis in SCC-25 cells in a time-dependent manner up to 24 h as determined by nuclear condensation and fragmentation of chromatin and DNA ladder formation. The present results indicate that the expression of Fas receptor and Fas ligand is negatively regulated by a protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in SCC-25 cells. Our results also suggest that Fas receptor and Fas ligand system might regulate apoptosis in SCC-25 cells in an autocrine fashion.

ヒト口腔へん平上皮癌細胞のオカだ酸誘導アポトーシスにおけるＦａｓレセプターおよびＦａｓリガンド発現の制御とｃａｓｐａｓｅ活性

Fas receptor is a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Binding of Fas ligand to Fas receptor leads to activation of the latter and the induction of intracellular signals that result in apoptotic cell death. In the previous studies, our group demonstrated that okadaic acid, protein phosphatase inhibitor, induced apoptosis in several cell lines including human oral squamous carcinoma cells. In the present study, RT-PCR and Western blot analysis were used to examine the expression of mRNA and proteins of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Expression of Fas receptor and Fas ligand mRNAs treated with okadaic acid was stimulated in dose- and time-dependent manners as judged by semiquantitative RT-PCR analysis, with the maximum expression level at 20 nM- and 8 h-treatment. Okadaic acid also stimulated the expression of Fas receptor and Fas ligand proteins in these cells in dose- and time-dependent manners as judged by Western blot analysis. These results indicate that the expression of Fas receptor and Fas ligand is negatively regulated by a protein phosphatase sensitive to okadaic acid. The activity of NF-κB was also stimulated by okadaic acid treatment indicating that expression of Fas receptor and Fas ligand system in SCC-25 cells is related in the activity of NF-κB.To investigate whether expression of Fas receptor and Fas ligand is involved in okadaic acid-induced apoptosis in SCC-25 cells, effects of okadaic acid on caspase-3 activities were examined. Caspase-3 activities increased after 12 h okadaic acid treatment and maximal activities were observed at 48 h treatment. Okadaic acid induced caspase-3 activities in a dose-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed in the okadaic acid-treated cells by using Hoechst 33342 staining and electron microscopic observation. Okadaic acid also induced DNA ladder formation in the treated cells with a time-dependent fashion from 12 h to 48 h. The present results indicate that the expression of Fas receptor and Fas ligand is involved in okadaic acid-induced apoptosis in SCC-25 cells. The present results also indicate that Fas receptor and Fas ligand system might regulate apoptosis in SCC-25 cells in an autocrine fashion.

Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase–polymerase chain reaction (RT–PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT–PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor–ligand system might regulate apoptosis in HSG cells.

Induction of intracellular signalling by cyclic glycerophosphates and their deoxy analogues.

Cyclic glycerophosphates can be formed by enzymic degradation of phospholipids. They have only recently attracted attention, and their physiological function is still obscure. In this study, we have searched for signalling functions of the natural 1,3-cyclic and 1,2-cyclic glycerophosphates, their deoxy analogues, and the phenyl esters of the 1,3-cyclic phosphates. Linear sn-glycerol 3-phosphate and glycerol 2-phosphate served as the control compounds. Each of the six-membered ring cyclic phosphates tested induced rapid intracellular tyrosine phosphorylation in CHO and NIH-3T3 cells when applied extracellularly at a concentration of 0.5-4 microM. The phosphorylated intracellular proteins had molecular masses of approximately 35 kDa, approximately 45 kDa, 60-70 kDa and approximately 120 kDa. The five-membered ring cyclic phosphates had a similar effect, but at an external concentration of 2-10 microM, while sn-glycerol 3-phosphate and glycerol 2-phosphate had no effect. The six-membered cyclic phosphates also induced rapid threonine phosphorylation in CHO cells of approximately 18-kDa, approximately 35-kDa, and approximately 38-kDa proteins. Further experiments indicated that the cyclic phosphates partition rapidly into the cell cytosol where they activate kinases, including mitogen-activated protein kinase. When their intracellular level increases, dephosphorylation presumably takes place. This pattern may account for the signalling profile of cyclic phosphates and suggests that they may take part in processes associated with cell differentiation.

CD69 and Regulatiof the Immune Function

CD69, also known as activation inducer molecule, very early activation antigen, MLR-3 and Leu-23, is a member of the natural killer (NK) cell gene complex family of signal transducing receptors. CD69 is as a type II transmembrane glycoprotein with a C-type lectin binding domain in the extracellular portion of the molecule.CD69 expression is induced in vitro on cells of most hematopoietic lineages, including T and B lymphocytes, NK cells, murine macrophages, neutrophils and eosinophils, while it is constitutively expressed on human monocytes, platelets and epidermal Langerhans cells.Although a specific ligand for CD69 has not been identified, its wide cellular distribution and the induction of intracellular signals upon CD69 crosslinking suggest a role for the receptor in the biology of hematopoietic cells. Moreover, certain results indicate that CD69 may be involved in the pathogenesis of such diseases as rheumatoid arthritis, chronic inflammatory liver diseases, mild asthma, and acquired immunodeficiency syndrome.

Induction of intracellular signals through binding of adhesive molecules. Activation of p125FAK tyrosine phosphorylation.

p125FAK tyrosine phosphorylation is a primary response to integrin stimulation. The first evidence that integrin occupancy by ligands induces tyrosine phosphorylation came from studies on the fibrinogen receptor αllbβ3 in platelets (). Further work showed that both in normal and transformed cells several different integrin receptors can trigger tyrosine phosphorylation of intracellular proteins following interactions with matrix proteins or integrin receptor clustering by specific antibodies ().